Methods and kits for quantifying the removal of mock virus particles from a purified solution

ABSTRACT

The present invention relates to a method of quantifying the amount of Mock Virus Particles (MVP) removed from a solution as a result of processing that solution through a purification technique. This method involves the steps of adding MVP to a solution, processing the solution through a purification technique, quantifying the amount of MVP removed from the solution. The present invention also relates to a kit that can be used in conjunction with the method. This kit will comprise at least one stock solution of MVP and at least one quantification solution.

SUMMARY OF THE INVENTION

The present invention relates to a method of quantifying the amount ofMock Virus Particles (MVP) removed from a solution as a result ofprocessing that solution through a purification technique. This methodinvolves the steps of adding MVP to a solution, processing the solutionthrough a purification technique, and then quantifying the amount of MVPremoved from the solution. The present invention also relates to a kitthat can be used in conjunction with the method. This kit preferablywill comprise at least one stock solution of MVP and at least onequantification solution.

BACKGROUND

Biopharmaceutical products, such as monoclonal antibodies, recombinantproteins, vaccines, blood derivatives and animal products carry a riskof transmitting infectious viruses (Burnouf, 2005; Aranha, 2011). Thisis due to either endogenous virus being present in the source materialused for biopharmaceutical manufacturing or the risk of exogenous“adventitious” virus contaminating a biopharmaceutical containingsolution during manufacturing (Kerr, 2010). As a result, manufacturersof biopharmaceutical products are required by international regulatoryagencies to incorporate sufficient virus clearance steps into theirmanufacturing processes and to validate these steps by providing robustviral clearance data (EMEA, 2008; EMEA, 2008; ICH, 1997; ICH, 998; FDA,1997).

To validate viral clearance, viral “spiking studies” are performedwhereby live virus is added to biopharmaceutical material and scaleddown purification process steps are performed (Darling, 2002). Thestep's ability to reduce virus is then analyzed by quantifying theremaining virus in solution via infectivity assay (TCID₅₀) orquantitative polymerase chain reaction techniques (Q-PCR). These studiesare usually conducted by third party contract labs due to the expertiseand additional safety measures required to propagate and quantify liveviral particles. As a result these studies are extremely expensive andlogistically difficult to conduct. In effect, process steps aretypically developed for months or years before they are evaluated forvirus removal efficacy. This practice increases regulatory risk as timeand money are spent developing process steps that may ultimately fail tosufficiently remove virus during regulatory enabling validation studies.Thus, there is a need for new and improved methods of determining virusremoval efficiency during purification processes development.

SUMMARY OF THE INVENTION

The present invention relates to a method of quantifying the amount ofMock Virus Particle (MVP) removed from a solution as a result ofprocessing the solution through a purification technique. The steps ofthe method include; adding MVP to a solution, processing the solutionthrough a purification technique, and quantifying the amount of MVPremoved from the solution. In a preferred embodiment, the solution towhich MVP is added contains a biologic of interest. In an even morepreferred embodiment, the biologic of interest is an antibody,non-antibody protein, vaccine, nucleic acid product, blood or plasmaderivative. In another even more preferred embodiment, the biologic ofinterest is produced by a cell culture process or a fermentation processwhich utilizes human cells, animal cells, plant cells, insect cells,hybridoma cells, yeast cells, or bacteria cells. In another even morepreferred embodiment, a biologic of interest present in the solution ispurified by way of processing of that solution through the purificationtechnique.

In another preferred embodiment, the purification technique thatprocesses a solution containing MVP is a chromatography, filtration,ultrafiltration, centrifugation, or viral inactivation technique. Inanother preferred embodiment, the quantity of MVP added to a solutionprior to processing that solution through a purification technique isgreater than the quantity of MVP in solution remaining after processing.

In a preferred embodiment, MVP comprises viral capsid protein, viralenvelope protein, or both a viral capsid and a viral envelope protein.In an even more preferred embodiment, the viral capsid or envelopeprotein is produced by a bacteria, yeast, plant, insect, and/or animaland/or human cell. In another even more preferred embodiment the viralcapsid or envelope protein is derived from a Parvoviridae orRetroviridae source. In another even more preferred embodiment, theviral capsid or envelope protein comprises a heterologous epitope. Inanother even more preferred embodiment, MVP contains in vitro nucleicacid.

In a preferred embodiment, quantifying the amount of MVP removed fromthe solution comprises the use of a quantification technique fordetermining the amount of MVP in a solution including Enzyme LinkedImmunosorbent Assay (ELISA), Polymerase Chain Reaction (PCR),nanoimaging, fluorescence, enzymatic, microscopy, spectrophotometry,Transmission Electron Microscopy (TEM), or western blot techniques. Inan even more preferred embodiment, the quantification technique uses anantibody capable of binding to a capsid protein epitope, an envelopeprotein epitope, or a heterologous epitope present on the surface of theMVP. In another even more preferred embodiment, the quantificationtechnique uses an antibody capable of binding to a linker molecule thatis bound to the MVP. In another even more preferred embodiment, thequantification technique uses a molecule bound to the MVP and anantibody capable of binding to the molecule or a primer capable ofbinding to a nucleic acid segment that is attached to the molecule. Inanother even more preferred embodiment, the quantification techniqueuses a primer capable of binding to an in vitro nucleic acid sequencecontained within the MW.

The present invention relates to a method whereby MVP is added to asolution, the solution is processed through a purification technique,and the amount of MVP removed from solution is quantified In a preferredembodiment, a second species of MVP is added to the solution, thesolution is processed through a purification technique, and the amountof the second species of MVP removed from solution is quantified In aneven more preferred embodiment, the first and second species of MVP areadded to a solution at the same time or sequentially. In another evenmore preferred embodiment, two or more additional species of MVP areadded to the solution.

The present invention also relates to a kit which comprises: at leastone container comprising a stock solution of MVP, and at least onecontainer comprising a quantification solution. In a preferredembodiment, the quantification solution comprises an antibody capable ofbinding to MVP or to a molecule which can be bound to MVP. In an evenmore preferred embodiment, the kit further comprises a solution of asecond antibody, capable of binding to the antibody which is capable ofbinding to MVP or to a molecule which can be bound to MVP. In anothereven more preferred embodiment, the antibody capable of binding to MVPis conjugated to an enzyme. In another even more preferred embodiment,the second antibody capable of binding to the antibody which is capableof binding to MVP or a molecule which can bind to MVP is conjugated toan enzyme. In another preferred embodiment, the kit further contains anELISA plate containing an immobilized antibody or molecule that can bindto MVP. In another preferred embodiment, the quantification solutioncomprises primers capable of binding to an in vitro nucleic acidsequence or a segment of nucleic acid bound to a molecule which can bebound to MVP. In another preferred embodiment, the kit contains anothercontainer comprising a solution of a molecule which can bind to MVP. Inanother preferred embodiment, the kit also contains additional reagentsfor performing ELISA or PCR techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments are illustrated by way of example and not limitation in thefigures of the accompanying drawings, in which like references indicatesimilar elements and in which:

FIG. 1: Purity of MMV MVP fractions

FIG. 2: Transmission electron microscopy image of MMV MVP stocksolution.

FIG. 3: Purity of heterologous epitope MMV MVP fractions.

FIG. 4: Transmission electron microscopy image of heterologous epitopeMMV MVP stock solution.

Mouse Minute Virus (MMV) MVP's were purified via methods referred to inExamples 1 and 2. To determine the purity of the Cesium Chloride densitygradient fractions, samples from each density fraction (lanes 1-13) werereduced and electrophoresed on a 4-12% polyacrylamide gel. Protein bandswere visualized through Commassie blue staining. A VP2 protein standard(alpha diagnostic cat#MVMVP25-R-10) was run in lane “S” for comparison(VP2 protein is expected to be 64 KDa) and a molecular weight markerprotein was run in lane “M”. In FIG. 1, MVP resulting from natural VP2protein formation was analyzed. Fractions 11-13 were pooled to form MVPstock solution. Based on staining results, the pooled stock solutioncontained MVP at a purity of >95%. In FIG. 3, MVP resulting fromrecombinant VP2 protein formation was analyzed. Fractions 11-13 werepooled to form MVP stock solution. Based on staining results, the pooledstock solution contained MVP at a purity of ˜90%.

MMV MVP stock solutions were produced via methods described in Examples1 and 2. In FIG. 2, TEM images were taken of MMV MVP stock solutionresulting from the assembly of 60 copies of natural (non-modified) VP2protein. In FIG. 4, TEM images were taken of MMV MVP stock solutionresulting from the assembly of 60 copies of recombinant VP2 proteins,each containing a heterologous epitope (strep II tag amino acidsequence). Images were captured after negative staining Two microlitersof each stock solution were placed onto separate formvar/carbon-coatedelectron microscope grids and allowed to air dry. After ten minutes,residual material was wicked from the grids. The grids were then fixedand stained by placing 20 microliters of 2.0% phosphotungstic acid(PTA), pH 7.0, onto each grid for one minute. The excess PTA was thenremoved, and the grids were examined, quantified and photographed usingan FEI Tecnai Spirit Twin microscope at a magnification of 165,000×.Results show concentrations of 3.06×10¹³ MMV MVP/ml of stock solution(FIG. 2) and 3.56×10¹³ heterologous epitope MMV MVP/ml of stock solution(FIG. 4).

DETAILED DESCRIPTION OF INVENTION

In the present invention, the term “mock virus particle (MVP)” refers toa non-infectious, non-replicating assembled unit comprised ofsynthetically produced (e.g. recombinantly expressed or chemicallysynthesized) viral capsid protein, viral envelope protein, or viralcapsid and envelope proteins. MVP's do not refer to virus particlesfound in nature, including, but not limited to live virus particles,virus particles found in nature that have naturally lost the ability tobe infectious, or virus particles that have lost the ability to beinfectious in vitro, such as “ultraviolet irradiated”, “heat-killed” or“heat-inactivated” viral particles. Thus, the synthetic nature of a MVPprovides their ability to be easily produced and used in a commercialsetting as compared to other forms of virus particles used in the art.The term “viral capsid protein” refers to a protein of any virus thatcomprises a shell around its genome. The term “viral envelope protein”refers to any viral protein that covers a capsid protein shell andbecomes part of the outer layer of a virus. Certain viral capsid andenvelope proteins are known to be prevalent to viruses within specificviral taxonomic families. MVPs can be produced from the capsid orenvelope proteins of these viral families resulting in units thatphysiochemically resemble specific viruses from within those families.However, assembled units of MVP lack genetic similarity to these viruses(MVP's may not contain any nucleic acid whatsoever). Examples of majorviral capsid and envelope proteins (common names of these proteinsreferred to in the art) and their associated viral family are listed inTable 1 below along with an example MVP which could assemble from one ormore of those proteins (Fauquet et. al, 2005).

TABLE 1 Known Capsid Protein Known Envelope Protein Virus FamilyExamples Examples Example MVP Parvoviridae VP1, VP2, VP3, VP4 None MouseMinute Virus - MVP Retroviridae MA, CA, NC (gag proteins) SU, TM, LP(env Xenotropic Murine proteins), Sag Leukemia Virus- Reoviridae μ1, μ2,μA, μB, λ1, λ2, λ3, λA, None Reovirus Type 3 - λB, λC, σ1, σ2, σ3, σA,σB, σC, MVP VP1, VP2, VP3, VP4, VP5, VP6, VP7, CSP, LPP, TP, P1, P2, P3,P5, P7, P8 Caliciviridae VP60, VP62, VP8.5, VP10, CP None FelineCalicivirus - MVP Tymoviridae CP None Physalis mottle-MVP HerpesviridaeVP5, VP1-3, VP23, VP26 gM, gB, gD, gL, gH, gC, Herpes simplex - VP19C,VP21, VP24, VP22, gE, gO, gI, gG gK, gJ, gN, MVP UL16, MCP, CP62, U56,U29, BMRF2, BDFL2, UL45H, U57 UL34, US9 Togaviridae CP E1, E2, E3Rubella -MVP Coronaviridae N S, M, E, HE Infectious bronchitis - MVPOrthomyxoviridae NP, PA, PB_(1,) PB₂ HA, NA, M₁, M₂, HEF, Influenza A -MVP GP, NB, BM₂, CM₂ Filoviridae NP, VP30, VP35, L GP, VP24, VP40Ebola - MVP Hepadnaviridae HBc L, M, S Hepatitis B - MVP ParamyxoviridaeNP, P M, F, HN, SH, G, H Human Parainfluenza 3-MVP Flaviviridae C M, E,prM, E^(ms), E1, E2 Bovine Viral Diarrhea-MVP Picornavirus VP1, VP2,VP3, VP4, Vpg, VP0 None Hepatitis A-MVP Polyomaviridae Vp1, Vp2, Vp3None Simian Virus 40 - MVP

In the present invention, a MVP unit assembles as the result ofrecombinantly expressing or chemically synthesizing viral capsid orviral envelope proteins in vitro. Preferably, viral capsid and envelopeproteins which assemble to form a MVP are expression products fromnaturally occurring viral protein nucleic acids sequences.Alternatively, they are expression products from viral protein nucleicacid sequences that have been altered, or modified, in vitro. In thepresent invention, protein products which are composed of altered ormodified amino acid sequences as a result of the expression of alteredor modified nucleic acid sequences are referred to as “recombinant”proteins. The act of altering or modifying naturally occurring viralprotein nucleic acid sequences to express recombinant viral capsid orenvelope proteins is well known in the art (see, for example, Gillock,1998). Preferably, recombinant MVP capsid or envelope proteins are 99.9%or more homologous to their natural viral protein sources, according tostandard protein based BLAST homology searches. Alternatively,recombinant capsid or envelope proteins of MVP are at least 50%, 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to their natural capsidand/or envelope protein sources, according to standard BLAST homologysearches.

Preferably, viral capsid or envelope proteins which assemble to formMVPs are produced by expressing their genes in bacteria, yeast, plant,insect, animal, or human cells. The act of producing these proteins inlieu of assembling a MVP is commonly known in the art (see for example,Makarova 2011). For example, natural or modified viral nucleic acidprotein sequences are first cloned into expression vectors. Preferably,expression vectors are yeast based expression vectors, bacterial basedexpression vectors, baculovirus based expression vectors, and/ormammalian based expression vectors, and/or plant-based expressionvectors. The expression vector is then made to transfect a cell.Preferably, cells that may be transfected include, but are not limitedto; bacteria, yeast, plant, insect, animal, mammal and/or human cells.Preferably, after the expression of natural or recombinant viral capsidor envelope proteins, the proteins spontaneously assemble into MVP.Alternatively, the assembly of MVP will not occur spontaneously. Inthese instances, the un-assembled protein containing solution could betreated with chemicals and/or proteins to increase the occurrence of MVPassembly. Alternatively, the un-assembled protein containing solutionwill be purified to increase the amount of capsid and or envelopeproteins in solution relative to other molecules in solution.

Preferably, the nucleic acid sequence expressed to produce a viralcapsid or envelope protein which assembles to form a MVP is derived froma Parvoviridae or Retroviridae genomic source. Examples of Parvoviridaederived nucleic acid sequence sources include, but are not limited tothe genomes of, Minute Virus of Mice (Mouse Minute Virus), CanineParvovirus, Feline Parvovirus, Porcine Parvovirus, B19 virus,Adeno-associated virus 1, Junonia coenia densovirus, Bombyx mori virus,and Aedes aegypti densovirus genomes. Examples of viral capsid proteinswhich could be produced and assembled to form MVP from these genomesinclude, but are not limited to, VP1, VP2, VP3, or VP4 proteins.Examples of Retroviridae derived nucleic acid protein sequence sourcesinclude, but are not limited to the genomes of, Avian ErythroblastosisVirus, Avian Leukosis Virus, Avian Myeloblastosis Virus, Avian SarcomaVirus, Avian Myelocytomatosis Virus, Esh Sarcoma Virus, Fujinami SarcomaVirus, Golden Pheasant Virus, Induced Leukemia Virus, Lymphoid LeukosisVirus, Myeloblastosis-associated Virus, Myelocytomatosis Virus,Rous-associated Virus, Ring-necked Pheasant Virus, Rous Sarcoma Virus,NK-24, SKV, Baboon Endogenous Virus, BEV, CCC, CERV-CI, CPC4, Corn SnakeRetrovirus, Chicken Syncytial Virus, Duck Infectious Anemia Virus, DeerKidney Virus, DPC4, Equine Dermal Fibrosarcoma Virus, Feline LeukemiaVirus, FeLV-AIDS, Feline Sarcoma Virus, Fr-MLV, Fr-SFFV, FS-1, GibbonApe Leukemia Virus, Hamster Leukemia Virus, Lymphoproliferative DiseaseVirus, Mink Cell Focus-inducing Virus, MAIDS, MDEV, Mink Leukemia Virus,Murine Leukemia Virus, MMCA, Murine Sarcoma Virus, Myeloid LeukemiaVirus, OMCA, PK-1S, R-35, RadLV, Rat Leukemia Virus, Ra-MCF, Ra-MLV,Ra-SFFV, Rat Sarcoma Virus, RDL14, Reticuloendotheliosis-associatedVirus, Spleen Focus-forming Virus, Simian Sarcoma Virus, Simian LymphomaVirus, Simian Myelogenous Leukemia Virus, Spleen Necrosis Virus, SimianSarcoma-associated Virus, Simian Sarcoma Virus, TRV4, Vand C-I, ViperRetrovirus, Woolly Monkey Virus, Woolly Monkey Leukemia Virus, BovineLeukemia Virus, BoLV, Human T-cell Leukemia Virus, Simian T-cellLeukemia Virus, STLVpan-p, Bovine Syncytial Virus, FelineSyncytium-forming Virus, Human Foamy Virus, Simian Foamy Virus, BovineImmunodeficiency Virus, Caprine Encephalitis-arthritis Virus, EquineInfectious Anemia Virus, Feline Immunodeficiency Virus, GoatLeukoencephalitis Virus, Human Immunodeficiency Virus, Jembrana,Maedi/visna Virus, Progressive Pneumonia Virus, Simian ImmunodeficiencyVirus, Mouse Mammary Tumor Virus, M432,M832, MNV, Mason-Pfizer MonkeyVirus, PMFV, P0-1-Lu, Squirrel Monkey Retrovirus, Simian Retrovirus,Jaagsiekte Retrovirus, Walleye Dermal Sarcoma Virus, Walleye DermalHyperplasia Virus, and Gypsy genomes. Examples of viral capsid andenvelope proteins which could be produced and assembled to form MVP fromthese Retroviridae genomes include, but are not limited to, gag proteins(MA, CA, NC), env proteins (SU, TM, LP), and Sag protein. Even morepreferably, Retroviridae derived protein sources include genomicsequences from mammalian cell-endogenous retroviruses and retroviruslike particles. Examples of mammalian cell-endogenous retroviruses andretrovirus like particles include, but are not limited to MurineLeukemia Viruses (Ab, AKT8, Cas-Br-E, Du5H MAIDS, FMCF-98, Fr, Graffi,Gross, LP-BMS, Ki, Mo, MPLV, NT40, PVC-211, Ra, RadLV, SL3-3, TRI-3,XMuLV) and Intracisternal A type particles. Examples of mammalian cellsthat may contain endogenous retrovirus or retrovirus like particlesinclude CHO, NS0, NS-1, Sp20Ag14, MH, BHK, and RH cells.

Preferably, a viral capsid or envelope protein assembles to form an MVPwhich displays an epitope(s) on its surface. In the present invention,an “epitope” is a specific sequence of amino acids displayed on theexterior surface of a MVP. Epitopes may be utilized to quantify theamount of MVP present in solution (and hence their removal fromsolution) without the need of infectivity assays, QPCR, or othercumbersome and expensive methods common to the art of quantifyinginfectious or non-infectious virus particle removal. In some instances,a recombinant viral capsid or envelope protein assembles to form an MVP.In these instances, the MVP may display a heterologous epitope(s) on itssurface. In the present invention, a “heterologous epitope” refers to anepitope which results from the expression of recombinant capsid orenvelope proteins. Likewise, an MVP can comprise a heterologous epitopewhen it is assembled from recombinant proteins. Examples of heterologousepitopes include but are not limited to, strep-tag (e.g. amino acidsequence WSHPQFEK (SEQ ID No:1)), flag tag (e.g. amino acid sequenceDYKDDDDK (SEQ ID No:2)) and His-tag (e.g. amino acid sequence HHHHHH(SEQ ID No:3)). Preferably, one copy of an epitope or heterologousepitope may be present per protein unit of that MVP. Alternatively,multiple copies of an epitope or heterologous epitope may be present perprotein unit of that MVP. Heterologous epitopes may enhance thesensitivity of quantification methods used to determine the amount ofMVP in a solution to levels beyond what is achievable for infectivityassays, QPCR assays or other assays currently common in the art.

Preferably, a MVP does not contain any nucleic acid. Alternatively, aMVP may contain a segment of in vitro nucleic acid. In the presentinvention, a “segment of in vitro nucleic acid” refers to a specificsequence of nucleic acid that is purposefully introduced to an MVPsolution as the particles are assembling so that the resulting MVPretains a copy of that sequence. Thus, unlike all other particles knownto the art, MVP's do not rely on inherited genetic material of a viralgenuine for quantifying their amount in solution. In the presentinvention, the term “inherited genetic material” refers to all naturallyencapsulated nucleic acids present in a replicating orreplication-deficient virus particle. For example, in the art thequantification of infectious virus particles involves either ameasurement of infectivity (the result of naturally encapsulated genomicnucleic acid expression) or QPCR (utilizing primers against theirnaturally encapsulated genomic nucleic acid) (Shi, 2004). Likewise, inthe art, the quantification of non-replicating endogenous retroviruslike particles involves QPCR utilizing primers against naturallyencapsulated genomic nucleic acid or quantitative product enhancedreverse transcriptase (Q-PERT) which measures viral reversetranscriptase activity (Zhang, 2008). Preferably, a segment of in vitronucleic acid may refer to a synthetically derived sequence of nucleicacid. Alternatively, a segment of in vitro nucleic acid may refer to anaturally derived sequence. Preferably, in the instance of a naturallyderived sequence, the length of the sequence is about 1% or less, about5% or less, about 10% or less, about 25%, about 30% or less, about 40%or less, about 50%, about 60% or less, about 70% or less, about 75% orless, about 90% or less, about 95% or less, or about 99% or less thanthe genome of the organism from which the sequence may have beenderived, No amount of in vitro nucleic acid will be sufficient to allowfor replication or infectivity of the MVP, as measured by methods commonin the art, One example of a method commonly used in the art to measureinfectivity is TCID₅₀. Unlike other virus particles common to the art,MVP's will thus have no risk of being or ever becoming infectious. Insome instances, an in vitro nucleic acid segment may be derived from aviral source. In other instances, an in vitro nucleic acid may bederived from non-viral sources. Examples of in vitro nucleic acidsources include but are not limited to, virus, bacteria, yeast, insect,animal, and/or human. Preferably, in instances where in vitro nucleicacid is derived from viral sources, the viral source may be the samesource from which the capsid or envelope proteins of the MVP werederived. Alternatively, in instances where in vitro nucleic acid isderived from viral sources, the viral source may be different from thesource from which the capsid or envelope proteins of the MVP werederived. Even more preferably, in either case, the 5′-end and the 3′-endof the in vitro nucleic acid segment can contain unique sequences thatare not present in the natural viral genome from which the sequence wasderived.

Preferably, after assembly, the MVP may be purified using methods knownin the art (Hernando, 2000), Moreover, the purity of MVP in its assemblysolution after purification can be such that less than 65% of allproteins in solution are non-MVP related, less than 55% of all proteinsin solution are non-MVP related, less than 45% of all proteins insolution are non-MVP related, less than 35% of all proteins in solutionare non-MVP related, less than 25% of all proteins in solution arenon-MVP related, less than 15% of all proteins in solution are non-MVPrelated, less than 5% of all proteins in solution are non-MVP related.In the present invention, the term “non-MVP related [proteins]” refersto all non-capsid and/or non-envelope proteins that do not assemble toform MVP. One example of a method to purify MVP is a sucrose densitygradient. Another example is centrifugation. Another example ischromatography. The purity of MVP in a stock solution can be determinedthrough methods common to the art including, but not limited to,Polyacrylamide Gel Electrophoresis (PAGE), high pressure liquidchromatography, mass spectroscopy, flow cytometry, ELISA, dynamic lightscattering, gel filtration, or ultracentrifugation. In some instances,after assembly, MVP may be introduced to and reacted against a linkermolecule so that the resulting MVP binds that linker molecule to itssurface. In the present invention, the term “linker molecule” refers toa synthetic polymer or natural polymer (such as a protein) that can becovalently or bound to another molecule.

As previously described, MVP are assembled from viral capsid or envelopeproteins. In the present invention, MVP are thus denoted according totheir viral protein source. For example, MVP assembled from the VP2protein (or recombinant versions of the MVP2 protein) of the MouseMinute Virus would be referred to as an “MMV MVP”. Another example wouldbe referring to a MVP assembled from env and/or gag proteins (orrecombinant versions of env and/or gag proteins) of the XenotropicMurine Leukemia Virus (XMuLV) as “XMuLV MVP”. In the present invention,MVP is preferably comprised of natural or recombinant viral proteinsproduced from Parvoviridae or Refroviridae nucleic acid sources.Alternatively, MVP is comprised of viral protein produced from nucleicacid sources of other virus families including, but not limited to,Caliciviridae, Reoviridae, Tymoviridae, Togaviridae, Herpesviridae,Coronaviridae, Orthomyxoviridae, Filoviridae, Hepadnaviridae,Paramyxoviridae, Flavivirdae, Picronaviridae, and/or Polyomaviridae.Preferably, an MVP is assembled from proteins derived from one viralsource. An example of MVP assembled from one viral source is MMV MVPassembled from a natural or recombinant MMV VP2 capsid protein.Alternatively, an MVP could assemble from protein derived from multipleviral sources. An example of MVP assembled from more than one viralprotein source is XMuLV MVP assembled from natural or recombinant XMuLVgag protein and natural or recombinant HIV env protein.

In the present invention, the term “species of MVP” refers to all MVP'scomprised of the same protein(s) and having the same copy number ofthose protein(s). For example, a species of MVP is all MVP's comprising60 copies of the MMV MVP2 protein. In a further preferred definition ofa species of MVP, the recombinant forms of a protein are to beconsidered the same as the natural protein from which it was derived.For example, MVP comprising 60 copies of recombinant MMV VP2 protein isthe same species as MVP comprising 60 copies of naturally derived MMVVP2 protein.

Preferably, the act of adding MVP to a solution refers to the additionof only one species of MVP to a solution. Alternatively, the act ofadding MVP to a solution refers to the addition of a second species ofMVP to a solution. Preferably, in these instances the first species andsecond species of MVP are added to solution at the same time.Alternatively, in these instances the first species and second speciesof MVP are added sequentially. One example of adding two species of MVPto a solution sequentially is adding MMV MVP to a solution first andthen XMuLV MVP to the same solution second. An example of adding twospecies of MVP to a solution at the same time is adding a solution thatcontains both MMV MVP and XMuLV MVP to another solution. In otherinstances, the act of adding MVP to a solution refers to the addition oftwo or more species of MVP to a solution.

Preferably, adding MVP to a solution refers to adding a volume ofsolution which contains a certain species of MVP to another solutionwhich does not contain that certain species of MVP. In the presentinvention the solution which does not contain a certain species of MVPuntil that species of MVP is added to it is referred to as a “processsolution”. For example, a solution of MMV MVP is added to a CHO cellsupernatant process solution which does not yet contain MMV MVP. Inanother example, a solution of XMuLV MVP is added to a CHO cellsupernatant process solution which contains MMV MVP but not yet XMuLVMVP. In the present invention, the solution containing MVP which isadded to the process solution can be referred to as a “stock solution ofMVP”, or “MVP stock solution”. Preferably, unlike stock solutions ofnon-infectious particles common to the art, stock solutions of MVP willhave known concentrations of MVP. For example, stock solutions of MVPcontained within the kit embodiments of this invention will include MVPconcentration information. Moreover, a stock solution of MVP has ahigher concentration of MVP than other non-infectious particles commonto the art. For example, MVP in a stock solution may be present atconcentrations of at least 1×10 MVP/ml, 1×10⁶ MVP/ml, 1×10⁷ MVP/ml,1×10⁸ MVP/ml, 1×10⁹ MVP/ml, 1×10¹⁰ MVP/ml, 1×10¹¹ MVP/ml, 1×10¹² MVP/ml,1×10¹³ MVP/ml, 1×10¹⁴ MVP/ml, 1×10¹⁵ MVP/ml, 1×10¹⁶ MVP/ml, or greater.In addition, MVP stock solutions will contain MVP at purities higherthan other non-infectious particles common to the art. For example,non-MVP related proteins in a stock solution of MVP may be less than 65%of all the proteins in the solution, less than 55% of all the proteinsin the solution, less than 45% of all the proteins in the solution, lessthan 35% of all the proteins in the solution, less than 25% of all theproteins in the solution, less than 15% of all the proteins in thesolution, less than 5% of all the proteins in the solution. Purity ofMVP in a stock solution can be determined through methods common to theart including, but not limited to, Polyacrylamide Gel Electrophoresis(PAGE), high pressure liquid chromatography, mass spectroscopy, flowcytometry, ELISA, dynamic light scattering, gel filtration, orultracentrifugation. Examples of producing stock solutions of MVP aredescribed in the examples section. Preferably, a stock solution of MVPcontains one species of MVP. One example of a MVP stock solutioncontaining one species of MVP is an MVP stock solution containing MMVMVP. Alternatively, a stock solution of MVP can contain multiple speciesof MVP. One example of a MVP stock solution containing multiple speciesof MVP is a stock solution containing MMV MVP and XMuLV MVP.

The quantity of MVP stock solution added to a process solution will varydepending on several factors including but not limited to, the volume ofprocess solution, the desired percent (v/v) of MVP stock solution in theprocess solution after addition, and the concentration of MVP in the MVPstock solution. Preferably, the volume of an MVP stock solution additionmay be in the order of milliliters or microliters. For example, thevolume of addition may be about 100 microliters or less, about 200microliters or less, about 500 microliters or less, about 1 milliliteror less, about 2 milliliters or less, about 5 milliliters or less, about10 milliliters or less, about 100 milliters or less, or about 1000milliliters or less. Alternatively, the volume of addition may beliters. For example, the volume of addition may be about 1 liter orless, about 2 liters or less, about 5 liters or less, or about 10 litersor less. Preferably, after addition, the percent of MVP stock solutionwithin a process solution may be about less than 1% (v/v) or less, about2% (v/v) or less, about 3% (v/v) or less, about 4% (v/v) or less, about5% (v/v) or less, about 10% (v/v) or less, about 25% (v/v) or less, orabout 50% (v/v) or less.

Preferably, the process solution contains a biologic of interest. In thepresent invention, the term “biologic of interest” refers to anymolecule produced by means of a biological process that may exhibittherapeutic potential. One example of a biological process in thepresent invention is cellular protein expression. In some cases,biologics of interest can be composed of sugars, proteins, nucleic acidsor complex combinations of these substances. In other cases, a biologicof interest may be living entities such as cells and/or tissues.Preferably, a biologic of interest is an antibody, a non-antibodyprotein, a vaccine, a nucleic acid, or a blood or plasma derivatives. Anexample of an antibody as a biologic of interest is Trastuzuman, whichis marketed under the trade name Herceptin™. Another example isRituximab, marketed under the trade name Rituxan™. Another example isbevacizumab, marketed under the trade name Avastin™. Examples of anon-antibody proteins as biologics of interest include, but are notlimited to, granulocyte colony stimulating factor (GCSF), a stem cellfactor, leptin, a hormone, a cytokine, a hentatopoietic factor, a growthfactor, an antiobesity factor, a trophic factor, an anti-inflammatoryfactor, a receptor, a soluble receptor, enzyme, and/or a variant, aderivative, or an analog of any of these proteins. Other preferredexamples of biologics of interest include but are not limited toinsulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroidstimulating hormone (TSH), luteinizing hormone (LH), folliclestimulating hormone (FSH), human chorionic gonadotropin (HCG), amotilin, an interferon (e.g., alpha, beta, or gamma), an interlenkin(e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11 and/or IL-12), tumor necrosis factor (TNF), tumor necrosisfactor-binding protein (TNF-bp), brain derived neurotrophic factor(BDNF), glial derived neurotrophic factor (GDNF), neurotrophic factor 3(NT3), a fibroblast growth factor (FGF), neurotrophic growth factor(NGF), a bone growth factor such as, for example, osteoprotegerin (OPG),an insulin-like growth factor (IGFs), macrophage colony stimulatingfactor (M-CSF), granulocyte macrophage colony stimulating factor(GM-CSF), megakaryocyte derived growth factor (MGDF), keratinocytegrowth factor (KGF), thrombopoietin platelet-derived growth factor(PGDF), a colony stimulating growth factor (CSFs), bone morphogeneticprotein (BMP), superoxide dismutase (SOD), tissue plasminogen activator(TPA), urokinase, streptokinase, or kallikrein, and/or a variant,derivative, or an analog of any of these proteins. One preferred exampleof a vaccine as biologic of interest is Recombivax HB. Another preferredexample of a vaccine is Gardasil. Another preferred example of a vaccineis Optaflu. Another preferred example is Cervarix. One preferred exampleof a nucleic acid as a biologic of interest is fomivirsen, which ismarketed under the trade name Vitravere™. Another preferred example of anucleic acid is mipomersen, which is marketed under the trade nameKynantro™. Another preferred example is Pegaptanib, which is marketedunder the trade name Macugen™. One preferred example of a blood orplasma derivate as a biologic of interest is albumin. Another preferredexample of a blood or plasma derivative is antihemophilic factor.Another preferred example is antihemophilic factor/von willebrand factorcomplex. Other preferred examples of biologics of interest in thepresent invention include but are not limited to anti-inhibitorcoagulant complex antithrombin (recombinant), c1 esterase inhibitor,coagulation factor, corifact, fibrin, fibrinogen, immune globulin,profilnine SD—factor IX complex, kcentra (Prothrombin ComplexConcentrate, Human), protein C concentrate (Human), thrombin, bonemarrow products, and embryonic fluid products.

Preferably, the biologic of interest in a process solution has beenproduced by a cell culture process or a fermentation process. In thepresent invention, the term “cell culture expression process” refers toa process by which cells are grown under controlled conditions toexpress a certain gene(s) (typically introduced in vitro). In thepresent invention, the term “fermentation expression process” refers toa process by which microorganisms are conditioned to grow and express acertain gene(s) (typically introduced in vitro). Preferably, cell linesfor cell culture or fermentation expression are of human animal, plant,insect, hybridoma, yeast, or bacteria origin. Examples of human celllines include but are not limited to, HeLa, NCI60, DU145, MCF-7, PC3,ARH-77, and/or HEK-293 cells. Examples of animal cell lines include butare not limited to, CHO, BHK, NSO, MDCK, Vero, GH3, PC12, and/or MC3T3cells. Examples of plant cell lines include but are not limited to,Tobacco BY-2 cells. Examples of insect cell lines include but are notlimited to, sf9, High Five, and/or C6/36 cells. Examples of yeastspecies from which yeast cell lines can be from include but are notlimited to, Saccharomyces cerevisiae and/or Pichia pastoris cells.Examples of bacteria species from which bacterial cell lines can be frominclude but are not limited to, Escherichia coli and/or Lactobacillus.Alternatively, cell lines for cell culture or fermentation expressionare of other origins. Examples of other cell lines include but are notlimited to, ZF4, AB9, and/or Xenopus A6 kidney epithelial cells.

In the present invention, the term “hybridoma” refers to a cell that isproduced in the laboratory from the fusion of an antibody-producinglymphocyte and a non-antibody-producing cancer cell, preferably amyeloma or lymphoma. Moreover, hybridomas of the present invention arecabable of proliferating and producing a continuous supply of specificmonoclonal antibody. Examples of hybridoma cell lines include but arenot limited to, RFT5, SP2/o cells, and/or HB54 cells.

In some instances, cell culture or fermentation processes which expressbiologics of interest co-express other biologics or molecules. In thepresent invention, all biologics or molecules that are co-expressedduring a cell culture or fermentation process that are not biologics ofinterest are referred to as “impurities”. Examples of impurities includebut are not limited to, host cell proteins (proteins expressed otherthan the biologic of interest), nucleic acids (besides a nucleic acidthat is a biologic of interest), charge variants of the biologic ofinterest, aggregate complexes, Beta-glucans, and/or virus. Additionally,impurities refer to all biologics, molecules, or chemicals that areadded to a solution containing a biologic of interest. Therefore, oneexample of an impurity is MVP after it has been added to a processsolution. In some instances a process solution may exist in the originalcell culture or fermentation expression solution along with alloriginating impurities. In other instances this solution may have beenpurified from its original state, prior to the addition of MVP, througha variety of techniques commonly known in the art as “purificationtechniques”. In the present invention, the term “purify” refers to anact of reducing the amount of impurities present in solution relative tothe amount of a non-impurity present in the same solution. Preferably, anon-impurity refers to biologic of interest present in the solution.Examples of purification techniques which may have purified the processsolution prior to the addition of MVP including but are not limited to,centrifugation, chromatography, filtration, precipitation,concentration, diafiltration, pasteurization, or viral inactivation. Insome instances, the solution may have been subjected to other techniquesor rigors including but not limited to, freezing, thawing, pHadjustment, and/or dilution prior to the addition of MVP.

The first embodiment of the present invention involves “processing thesolution through a purification technique”. In this step of the method,the term “solution” refers to the process solution after a quantity ofMVP stock solution has been added to it. Preferably, this solutioncontains a biologic of interest. As previously mentioned, non-biologicsof interest, “impurities” may also be present, including MVP. During thefirst embodiment of the present invention, this solution is “process[ed]. . . through a purification technique”. In the present invention, theterm “purification technique” refers to techniques which “purify” thesolution, that is, techniques which reduce the amount of impuritiespresent in solution relative to the amount of a non-impurity present inthe same solution. Preferably, a non-impurity refers to a biologic ofinterest. Thus, a further preferred embodiment of the present inventionis to purify a biologic of interest present in the process solutionthrough an act of processing that solution through a purificationtechnique.

Preferably, the purification technique used to process the processsolution is a chromatography, filtration, ultrafiltration,centrifugation, or viral inactivation technique. In the presentinvention, chromatography, filtration, ultrafiltration, orcentrifugation can be referred to as “separation techniques”. Separationtechniques are methods of mass transfer that distribute the constituentsof a solution into two or more distinct solutions. Separation techniquesare carried out based on differences in physical and chemical propertiesbetween the various components of a solution, including but not limitedto, size, shape, mass, and/or chemical affinity. Examples of separationtechniques include but are not limited to, affinity chromatography,ion-exchange chromatography, hydrophobic interaction chromatography,reverse phase chromatography, mixed mode chromatography, depthfiltration, size based filtration (including nanofiltration, sterilefiltration, or ultrafiltration), and centrifugation. Viral inactivationtechniques refer to any method aimed at reducing the abilities of virusto retain its proper structure or replicate. Examples of viralinactivation techniques include exposure to solvent and detergent orchemical treatments, low pH, heat, or ultraviolet radiation.

The first embodiment of the present invention involves “processing thesolution through a purification technique”. In the present invention,the term “processing” refers to the act of physically performing apurification technique. Different physical acts of processing aseparation technique include, but are not limited to, pumping, applyingdirect pressure, centrifugation, gravity, or shaking. In some instances,more than one way of processing may apply for one separation technique,depending on the format of the separation technique. For example, theformat of an ion exchange chromatography technique may be a packedcolumn, filter, or 96 well plate. Therefore the act of processing thision exchange chromatography technique may consist of pumping, applyingpressure, centrifugating, gravity, and/or shaking Different physicalacts of processing a viral inactivation technique include, but are notlimited to, adding organic solvents, detergents or acidic solutions,microwaving, exposing to UV light, immersion in hot water bath,pasteurization, or steam treatment.

In some cases, processing a solution through a separation techniquereduces the amount impurities in solution and is therefore said to“purify” the solution. After addition of MVP to a process solution, MVPis considered an impurity. Preferably, the quantity of MVP present inthe process solution is reduced through the act of processing, ascompared to the quantity of MVP present before such processing.Alternatively, the quantity of MVP present in the process solution isnot reduced through processing. The ability of a purification techniqueto reduce the amounts of impurities in a solution relies on a set ofparameters, or “variable inputs”, that someone skilled in the artutilizes to process. Examples of variable inputs include but are notlimited to; pH, conductivity, and temperature of the solution to beprocessed. Other examples of variable inputs include but are not limitedto, pressure applied, exposure time, or flow rate of a solution. Anotherexample is the concentration of constituents in the solution. Otherexamples are pH, conductivity, or chemical composition of buffers usedto process a solution. Another example is the criteria used forcollecting the process solution during or after the act of processing.Thus, the set of parameters utilized to process a solution through apurification technique impacts the effectiveness of the techniques'ability to reduce impurities (such as MVP) relative to non impurities(such as biologics of interest).

In some cases, an effective criteria for collecting process solutionduring or after processing is employed which results in fewerimpurities. Once the act of processing has begun, the methodology ofcollecting process solution(s) relies on someone skilled in the art. Inthe present invention, a process solution which has been collected sincethe act of processing has begun is referred to as “process collections”.Examples of methodologies used to collect process collection during apurification technique include but are not limited to, light absorbancedetection and fixed volume. Preferably, someone skilled in the art willutilize an effective collection criteria during or after processing sothat a process collection contains less impurities that the processsolution did before processing. Even more preferably, a collectioncriteria is utilized so that a process collection contains less MVP thanthe process solution prior to processing.

In some cases, distinct process collections are collected duringprocessing. One example of how a distinct process collection iscollected during processing is a collection of column effluent duringthe loading phase of a chromatography separation technique. Anotherexample would be collecting the column effluent during the wash phase ofa chromatography separation technique. Another example would becollecting the column effluent during the elution phase of achromatography separation technique. Another example would be collectingthe filtrate of a filter. Another example would be collecting thesolution during low pH titration. Another example would be collectingthe solution during exposure to UV light or chemical treatment.Alternatively, distinct process collections may be collected afterprocessing. One example of how distinct processed solutions arecollected after processing is by collecting the column effluent duringthe strip phase of a chromatography separation technique. Anotherexample would be collecting the solution after low pH titration followedby an increase in pH and filtration. Another example would be collectingthe solution after exposure to UV light or chemical treatment.

The first embodiment of the present invention involves “quantifying theamount of MVP removed from the solution”. In this specific embodiment,the act of “quantifying” refers to the means by which someone skilled inthe art mathematically calculates the amount of MVP removed fromprocessing the solution. Preferably, this value may be expressed as alog reduction value (LRV). Alternatively this value may be expressed asa molarity (mol/L), in total grams of MVP, and/or in total molecules ofMVP. Preferably, someone skilled in the art could mathematicallycalculate the amount of MVP removed from the solution by an equationrelating the amount of MVP remaining in solution after processing to theamount of MVP in solution prior to processing. Preferably, the quantityof MVP present in solution prior to processing is known by multiplyingthe volume of an MVP stock solution added to a process solution by theMVP concentration of that MVP stock solution. Even more preferably, thequantity of MVP present in solution prior to processing could bedetermined empirically. Likewise, preferably, the quantity of MVPremaining in a process collection could be determined empirically.Different techniques can be utilized for determining the amount of MVPpresent in solution empirically. In this present invention, thesetechniques will be referred to as “quantification techniques”. Preferredexamples of how to quantify the amount of MVP removed from solution areshown in the examples section.

Preferably, “quantification techniques” used for empirically determiningthe amount of MVP in a solution include ELISA, PCR, nanoimaging,fluorescence, enzymatic, microscopy, spectrophotometry, transmissionelectron microscopy (TEM), and western blot techniques. In thisembodiment of the present invention, the “solution” from which theamount of MVP is being determined, refers to a process solution after anaddition of MVP, a process collection(s), or aliquots taken of either.In this embodiment, the solution can be referred to as “an MVPcontaining solution”. Preferably, when performing a quantificationtechnique, a solution which contains an agent capable of binding to aMVP or to a molecule attached to a MVP, is added to an MVP-containingsolution. One example of such an agent is an antibody. Alternatively,when performing a quantification technique, a solution which containsPCR primers capable of binding to an in vitro nucleic acid or to anucleic acid sequence bound to a molecule which can be first bound to aMVP, is added to an MVP containing solution. In the present inventionthe solution containing an agent or PCR primer is referred to as “aquantification solution”.

Preferably, during the act of quantifying the amount of MVP in asolution, a serial dilution of MVP in process solution will be made andanalyzed via a quantification technique. Preferably, the data from suchanalysis will relate the quantity of MVP in a solution to a signalreceived as a result of the quantification technique. Examples ofsignals received as part of a quantification technique include, but arenot limited to, Ocular Density (OD), Absorbance Units, pRNA copies perml, pDNA, copies per ml, RNA copies per ml, DNA copies per ml, or unitsof reverse transcriptase activity. Even more preferably, a line of bestfit will be used in conjunction with the data to relate quantificationtechnique signals generated by unknown quantities of MVP to signalsgenerated by known quantities. Examples of making and using serialdilutions to quantify the amount of MVP in solution in lieu ofquantifying MVP removal from a solution are shown in the examplessection.

In some cases a MVP will be composed of natural or recombinant viralcapsid or envelope protein and display epitopes or heterologous epitopeson its surface. Preferably, in these instances, antibodies that bind tothese epitopes or heterologous epitopes can be utilized during aquantification technique to determine the amount of MVP present insolution. One example of how an antibody binding to an epitope displayedon an MVP could be utilized to determine the amount of MVP is by addinganti-VP2 antibody directed against a natural or recombinant VP2 capsidprotein to an MMV MVP containing solution during an ELISA quantificationtechnique. An example of how an antibody binding to a heterologousepitope displayed on an MVP could be utilized to determine the amount ofMVP is by adding an anti-his antibody directed against a his tag(present as a heterologous epitope on the surface of the MVP) to an MVPcontaining solution during an ELISA quantification technique.Preferably, antibodies known in the art to be capable of binding toepitopes or heterologous epitopes contained by a MVP can be the agentused in a quantification solution. Even more preferably, novelantibodies made by using MVP as an immunogen in an organism can be theagent used in a quantification solution. Thus surprisingly sensitivequantification measurements and MVP removal calculations can be achievedthrough non-genetic material based techniques common to the art.

In other cases a MVP will be bound to a linker molecule. Preferably, inthese instances, antibodies that bind to linker molecules can be addedto an MVP containing solution during a quantification technique todetermine the amount of MVP present in solution. In other cases, asolution containing a molecule can be added to an MVP containingsolution prior to addition of a quantification solution. In the presentinvention, the term “molecule” refers to a natural or artificial smallmolecule or protein which has an affinity for a MVP.

In some cases, molecules are added to MVP-containing solution to form aMVP-molecule complex. Preferably, in these cases, molecules do not havenucleic acid sequences attached and displayed on their surface. In othercases, molecules may have nucleic acid sequences attached and displayedon their surface. Preferably, in instances when a solution containing amolecule is added to an MVP containing solution, a quantificationsolution is then added to determine the amount of MVP present insolution via a quantification technique. One example of how the amountof MVP present in solution is determined by adding a solution containinga molecule is by first adding a solution containing streptactin to asolution containing strep tag-MVP (MVP comprising a strep tagheterologous epitope) and then using an anti-streptactin antibody todetermine the amount of MVP with an ELISA quantification technique.Another example is by first adding a solution of nucleic acid-conjugatedstreptactin (streptactin containing an attached segment of nucleic acid)to a MVP containing solution and then using primers directed against thesegment of nucleic acid to determine the amount of MVP with a PCRquantification technique.

In some cases, a MVP will contain within its structure a segment of invitro nucleic acid. Preferably, in these instances, a quantificationsolution containing primers that bind to in vitro nucleic acid containedwithin the MVP can be utilized to determine the amount of MVP with a PCRquantification technique. In some instances methods of enhancing signalsgenerated by quantification techniques common to the art may be used.One example of a method of enhancing signals generated by quantificationtechniques involves metal enhanced luminescence.

Preferably, quantifying the amount of MVP removed refers to one speciesof MVP. Preferably, in these instances, one species of MVP was added toa process solution which was processed through a purification technique.Alternatively, in instances where two or more species of MVP are addedto a process solution which is processed through a purificationtechnique, quantifying the amount of MVP removed may refer to multiplespecies of MVP. Preferably, in these instances, the same quantificationtechniques may be used in determining the amounts of multiple species ofMVP in a solution. One example of using the same quantificationtechnique in determining the amounts of multiple species of MVP in asolution is using an anti-VP2 antibody which binds to MMV MVP and ananti-env antibody which binds to XMuLV MVP in separate ELISAquantification techniques. Alternatively, different quantificationtechniques may be used in determining the amounts of multiple species ofMVP in a solution One example of using different quantificationtechniques is using an ELISA based technique to determine the amount ofMMV MVP in solution and using a PCR technique used to determine XMuLVMVP in the same solution.

Preferably, the steps of adding MVP to a solution, processing thesolution through a purification technique, and quantifying the amount ofMVP removed from solution are to be performed sequentially andun-interrupted. Alternatively, additional steps may be includedaccording to rational experimental design. Examples of additional stepsthat may be included according to rational experimental design includebut are not limited to, further purifying the stock solution of MVPprior to adding it to a process solution (via filtering, chromatography,or other techniques), performing dialysis or diafiltration on the stocksolution of MVP prior to adding it to a process solution, adding anon-MVP solution to the process solution before or after the addition toMVP. An example of a non-MVP solution is a cell culture suspension oflive virus preparation not containing virus. Examples of otheradditional steps may include taking an aliquot of the process solutionafter addition of MVP but prior to processing through a purificationtechnique, centrifuging or diluting a process collection or an aliquotof a processed collection prior to performing a quantificationtechnique, and/or freezing and thawing the aliquot taken for aquantification technique prior to performing the quantificationtechnique.

Thus, one embodiment of the present invention is a method of quantifyingthe amount of MVP removed from a solution. Another embodiment of theinvention is a kit used for executing the method. Preferably, the kitwill contain one container comprising a stock solution of a singlespecies of MVP and one container comprising a quantification solution.Alternatively, the kit will contain one container comprising a stocksolution of MVP which contains multiple species of MVP. Preferably, inthis instance, the kit will also contain multiple containers ofquantification solution for empirically determining the amount of eachspecies of MVP present in the stock solution bottle. In instances wherethe kit contains multiple stock solution bottles of MVP (containingdifferent species of MVP), the kit will also contain multiplequantification solution containers for determining the amount of thosespecies of MVP.

In the present invention, the “container comprising a stock solution ofMVP refers to a bottle contained within the kit that contains a stocksolution of MVP at a known concentration (of MVP). Moreover, the MVP ina stock solution container is present at a concentration and purity thatexceeds the concentration and purity levels of other non-infectiousparticles common to the art. For example, the concentration of an MVP ina stock solution container may be at least 1×10⁵ MVP/ml, 1×10⁶ MVP/ml,1×10⁷ MVP/ml, 1×10⁸ MVP/ml, 1×10⁹ MVP/ml, 1×10¹⁰ MVP/ml, 1×10¹¹ MVP/ml,1×10¹² MVP/ml, 1×10¹³ MVP/ml, 1×10¹⁴ MVP/ml, 1×10¹⁵ MVP/ml, 1×10¹⁶MVP/ml, or greater and the non-MVP related proteins may be present atlevels less than 65% of all the proteins in the solution, less than 55%of all the proteins in the solution, less than 45% of all the proteinsin the solution, less than 35% of all the proteins in the solution, lessthan 25% of all the proteins in the solution, less than 15% of all theproteins in the solution, less than 5% of all the proteins in thesolution. Preferably, the MVP in a stock solution bottle may be in theoriginal cell culture or fermentation based expression solution fromwhich the MVP assembled. Even more preferably, the MVP in a stocksolution is purified so that concentrations of cellular non-MVP relatedproteins, nucleic acids, or lipids in solution are reduced as comparedto the original expression solution from which the MVP assembled. Evenmore preferably, the MVP in a stock solution bottle is highly purifiedfrom the original expression solution from which the MVP assembled. Insome instances, a stock solution of MVP may contain added buffercomponents. Preferably, a single stock solution bottle of MVP containsonly one specific species of MVP. Alternatively, a single stock solutionbottle contains multiple species of MVP.

In the present invention, the “container comprising a quantificationsolution” refers to a bottle contained within the kit that comprises aquantification solution containing an agent capable of binding to a MVP,an in vitro nucleic acid, a molecule attached to MVP, or a nucleic acidsequence bound to that molecule. Preferably, a quantification bottlecomprises a quantification solution containing an antibody capable ofbinding to MVP or to a molecule which can be bound to an MVP. Oneexample of a quantification bottle comprising a quantification solutioncontaining an antibody is a quantification solution bottle, comprising aquantification solution containing anti-VP2 antibody which can beutilized during an ELISA quantification technique to determine theamount of MMV MVP in a solution. In these instances, the antibody maybind to epitopes or heterologous epitopes present on the MVP or epitopespresent on the molecule. In an even further preferred embodiment of theinvention, the kit also contains a solution of a secondary antibodycapable of binding to the primary antibody which binds to MVP or amolecule bound to MVP. Preferably, the solution of secondary antibody isadded during the execution of a quantification technique after theaddition of antibody capable of binding to MVP or molecule.

In some instances, an antibody which can bind to an MVP or a moleculebound to an MVP is not conjugated to an enzyme. Alternatively, in afurther preferred embodiment of the present invention, an antibodycontained in a quantification solution which is capable of binding toMVP or to a molecule bound to a MVP is conjugated to an enzyme. Examplesof enzymes which can be conjugated to antibodies are horse radishperoxidase (HRP) and alkaline phosphatase. Likewise, in another furtherpreferred embodiment, a secondary antibody which binds to an antibodycapable of binding to an MVP or a molecule bound to an MVP is conjugatedto an enzyme.

In a further preferred embodiment of the invention, the kit furthercomprises an ELISA plate containing an immobilized antibody or moleculethat binds to MVP. Preferably, the plate contains 96 wells.Alternatively the plate may contain less than 96 wells. Preferably, theimmobilized antibody or molecule contained in the ELISA plate bind tothe MVP contained within the MVP stock solution of the same kit.

Alternatively, in another further preferred embodiment, thequantification bottle comprises a quantification solution containingprimers capable of binding to an in vitro nucleic acid sequence or to asegment of nucleic acid bound to a molecule which can be bound to a MVP.Preferably, in these instances, a quantification solution bottle maycontain PCR primers specific to a segment of in vitro nucleic acidcontained within MVP. Alternatively, in these instances, aquantification solution bottle may contain PCR primers specific to asegment of nucleic acid that is adhered to a molecule that may be firstbound to a MVP during the step of quantification.

in a further preferred embodiment of the present invention, the kitfurther comprises a solution of a molecule which can bind to MVP. Oneexample of a solution of a molecule is a solution containingstreptavidin. Another example of a solution of a molecule is a solutioncontaining streptavidin displaying a short nucleic acid sequence.Preferably this solution will be added during execution of aquantification technique prior to the addition of a quantificationsolution.

In a further preferred embodiment of the invention, additional reagentsfor performing ELISA or PCR are included in the kit. Examples ofadditional reagents for performing ELISA or PCR include common buffers,enzymes, or molecules common to the art.

Although embodiments have been described with reference to specificexample embodiments, it will be evident that various modifications andchanges can be made to these example embodiments without departing fromthe broader spirit and scope of the present application. Accordingly,the specification and drawings are to be regarded in an illustrativerather than a restrictive sense.

EXAMPLES Example 1 Cloning, Expressing and Purifying Mouse Minute Virus(MMV) Mock Virus Particles (MVP) to Produce a Stock Solution

Mouse minute virus (MMV) is a single-stranded DNA containing virusbelonging to the family Parvoviridae that infects vertebrate hosts. Themouse minute virus capsid protein gene, VP2, can be cloned and expressedusing a baculovirus expression system to generate MVP (Hernando, 2000).In order to clone and express MMV MVP, the capsid protein gene VP2 wassynthesized from a published MMV VP2 sequence template (GenBankJ02275.1, nucleotides 2794-4557, SEQ ID No.4). Certain codons wereoptimized during this synthesis to increase the efficiency oftranslation (SEQ ID No. 5). The resulting amino acid sequence (SEQ IDNo. 6) was 100% homologous to the published VP2 sequence (GenBankAAA67114.1, SEQ ID No. 7). The gene was inserted into a cloning vector,pUC57 from which it was then subcloned into a pFastBac expressionvector. This vector was then used to transform DH10Bac cells. Afterscreening for positive clones, bacmid DNA was used to transfect Sf9cells. Recombinant baculovirus carrying MMV VP2 gene was collected fromSf9 cell culture supernatant. The original recombinant baculovirus stockwas then amplified and was collected at 4 days post infection. Thisstock was cultivated in Grace's medium supplemented with 10% FBS and wasthen used to transfect Sf9 cells at a multiplicity of infection of 4.0.Cells were then harvested at 3 days post-infection and resuspended inlysis buffer. This suspension was then frozen and thawed 3 times.Soluble lysate was recovered by centrifugation and then purification ofthe resulting MVPs was performed following a published protocol(Hernando, 2000). The purity of MVP after Cesium Chloride densitygradient fractionization was determined through SDS-PAGE with Coomassieblue staining (FIG. 1) and western blot analysis (not shown). Based onthe results, fractions were pooled to form MMV MVP stock solution. Avisualization of MMV MVP stock solution and a concentrationdetermination were made through transmission electron microscopy withnegative staining (FIG. 2).

Example 2 Cloning, Expressing and Purifying Heterologous Epitope MMV MVPto Produce a Stock Solution

MMV MW's can be made to display heterologous epitope(s) on the surfaceof its structure and could thus be used as a target for MVPquantification. In order to clone MMV MVP displaying a heterologousepitope, the natural nucleotide sequence of the MMV VP2 gene was firstsynthesized (using GenBank J02275.1, nucleotides 2794-4557, SEQ ID No.4as a template) while optimizing certain codons to increase theefficiency of translation (SEQ ID No. 5). This sequence then underwentmutagenesis (SEQ ID No. 8), at amino acid position 2, resulting in anamino acid sequence which included the insertion of a 10 amino acidsequence containing a strep II tag (SEQ ID No. 9). The same methods andprocedures were then utilized from example 1 to clone, express, purifyand produce heterologous epitope (strep II tag) containing MVP stocksolution. The purity of MVP after Cesium Chloride density gradientfractionization was determined through SDS-PAGE with Coomassie bluestaining (FIG. 3) and western blot analysis (not shown). Based on theresults, fractions were pooled to form heterologous epitope MMV MVPstock solution. A visualization of the resulting stock solution and aconcentration determination were made through transmission electronmicroscopy with negative staining (FIG. 4). Further validation that theresulting MVP displayed strep II tag was made through an ELISA assaywhich utilized streptactin and mAb against the tag (data not shown).

Example 3 Cloning, Expressing and Purifying a Xentropic Murine LeukemiaVirus (XMuLV) MVP to Produce a Stock Solution

It has been shown previously that the infection of cells with Ad5vectors that co-express XMRV env and gag genes lead to the production ofnon-infectious particles (Makarova, 2011). First, the XMulV gag and envgenes can be custom synthesized using the published sequences of thegenes as templates (GenBank accession number JF908817.1, nucleotides546-2156, SEQ ID No. 10, and accession number K02730.1, nucleotides291-2225, SEQ ID No. 11, respectively). Then the nucleic acid sequencescould be cloned into pUC57 vectors. Next, the env sequence can besub-cloned into CMV-driven expression cassette of pDP1 Shuttle vectorand the gag sequence could be cloned into the MCMV-driven expressioncassette of the same vector resulting in pDP1-XMuLVenvgag. ThepDP1-XMuLVenvgag plasmid could then be linearized and mixed with thepAdEasy-1 plasmid before co-transfecting 293-AD cells to producerecombinant Ad5-XMuLV. The recombinant adenovirus could be purified bydouble centrifugation on cesium chloride gradients. To produce an MVPstock solution, Mv1Lu cells can be infected with Ads-XMuLV for virusabsorption. Culture media could then be collected after 48 hours ofinfection, passed through a 0.45-mm filter, and concentrated/purified byultracentrifugation through a sucrose gradient.

Example 4 Cloning, Xpressing and Purifying XMuLV MVP ContainingHeterologous Epitopes to Produce Stock Solution

XMuLV MVP's can be made to contain heterologous epitope(s) on thesurface of its structure through methods discussed in Suomalainen etal., 1994. Alternatively, the nucleotide sequences of either the XMuLVgag and/or env gene (GenBank accession number JF908817.1, nucleotides546-2156, SEQ ID No. 10, and accession number K02730.1, nucleotides291-2225, SEQ ID No. 11, respectively) could be synthesized to includethe sequence for a heterologous tag such as, but not restricted to,astrep-tag (amino acid sequence WSHPQFEK (SEQ ID No:1)), a Flag tag(amino acid sequence DYKDDDDK (SEQ ID No:2)) or a His-tag (amino acidsequence HHHHHH (SEQ ID No:3)). Cloning, expression, and purificationcould occur as described in Example 3 above.

Example 5 Assembling XMuLV MVP Containing a Nucleic Acid

In order to generate XMuLV MVP containing a piece of nucleic acid, XMuLVgag- and/or env protein can first be expressed from mammalian cell asdescribed in the example 4. In vitro assembly of XMuLV gag and/or envprotein to include nucleic acid, which could be DNA or RNA, can then becarried out following published protocols (Gross et al., 1997; Yu etal., 2001).

Example 6 Quantifying the Removal of MMV MVP from a mAb ContainingSolution After Processing Through an Anion Exchange Column

NS0 harvest cell culture fluid containing a monoclonal antibody (mAb1)was thawed from storage −80 degrees Celsius. The material was titratedto a pH of 7.5 with 1 M Tris and then filtered through a 0.22 μm filter.One hundred microliters of MMV MVP stock solution (at a concentration of1×10⁹ MVP/ml) comprising 60 copies of VP2 capsid protein displaying aheterologous strep II tag epitope was added to 10 mls of the mAb1process solution (1% v/v addition). The process solution thus had aconcentration of 9.9 x 10⁶ MVP/ml ((0.1 ml×1×10⁹ MVP/ml)/10.1 mls) Next,a 0.66 cm×2 cm Q Sepharose Fast Flow column was packed to vendorrecommended specifications (GE healthcare) and equilibrated with 50 mMTris-HCl, 50 mM NaCl (pH 7.5) at a flow rate of 60 cm/hr using an AKTAexplorer. After equilibration, the 10.1 mls of process solutioncontaining MVP was loaded through the column at 60 cm/hr. Processcollections were taken as the UV₂₈₀ trace indicated flow through ofprotein. 200 ul samples of the collections were taken.

An ELISA quantification technique was then performed to quantify theamount of MVP removed in each of the process collections frompurification processing. Microtiter wells were first coated with rabbitpolyclonal anti-MMV VP2 antibody (Alpha Diagnostic, Cat#MVMVP21-S). 50uls of each of the process collection samples were added to the coatedwells, incubated for 1 hour and washed three times with 1× PhosphateBuffer Saline (PBS). In addition a serial dilution of the MMV MVP stocksolution was made in the original process material resulting in MVPconcentration of 1×10⁸ MVP/ml, 1×10⁶ MVP/ml, and 1×10⁴ MVP/ml. 50 uls ofeach dilution were also added to coated wells, incubated, and washed.Rabbit polyclonal anti-MMV VP2 antibody was then added to each well,incubated for 1 hour and washed 3 times with 1×PBS. HRP-conjugated antirabbit antibody (1:500) was then added, incubated for an hour, andwashed 3 times with 1×PBS. TMB substrate solution was added and thereaction was stopped by addition of stop solution. Optical Density (OD)was measured at 450 nm. The OD₄₅₀ results are shown in Table 2 below.

TABLE 22 OD₄₅₀ measurements from MMV MVP removal study Sample OD₄₅₀ MVPDilution 1 (1 × 10⁸ MVP/ml) 1.1 MVP Dilution 2 (1 × 10⁶ MVP/ml) 0.9 MVPDilution 3 (1 × 10⁴ MVP/ml) 0.52 MVP Dilution control (process solution)0.03 Process collection 1 0.04 Process collection 2 0.01

A line of best fit was established with the three dilution samples ofknown concentration (relating OD₄₅₀ and MVP concentration). The equationfor this line was:

Y=0.0525ln(x)+0.1235

By plugging in the OD₄₅₀ results from the two process collections, theMVP concentrations in those samples were determined. It was thus foundempirically that 0 MVP/ml remained in either process collection. Sincethe limit of detection in this ELISA assay is unknown, the limit isassumed to be lowest concentration of MVP tested (1×10⁴ MVP/ml). The logreduction value of MMV MVP was calculated from the known amount of MVPin the process solution and the empirically determined amount of MVPremaining in the process collections. This value, ≥9.9×10², is thereforethe quantity of MVP removed from solution by way of processing thatsolution through a purification technique.

Example 7 Quantifying the Removal of XMuLV MVP from a Mab ContainingSolution After Processing Through a Parvovirus Filter

A solution from a biotechnology process that contains a monoclonalantibody can be purified through protein affinity and ion exchangechromatography columns using methods familiar to the art. The solutioncould be frozen at −80 degrees Celsius for several months and thenthawed and filtered through a 0.22 um filter. 12.5 mls of a XMuLV MVPstock solution could then be pippetted into 250 mls of the filteredprocess solution (5% spike v/v). A 1 ml sample of this MVP added processsolution would be taken for later quantification. The process solution(now containing XMuLV MVP) would then be pressurized through a Vproparvovirus filter at 30 psi and the filtrate would be collected. A 1 mlsample of this filtrate (process collection) would be taken.

A serial dilution of XMuLV MVP in process solution could be preparedwith dilutions of MVP at concentrations of 1×10⁹, 1×10⁷, 1×10⁵, and1×10³ MVPs/ml of process solution. 50 uls of each dilution could then beadded to microtiter wells coated with antibody against an XMuLV envepitope. The wells would then be incubated for an hour and washed threetimes with 1×PBS buffer. Next, a HRP conjugated antibody against adifferent env epitope could be added to each well, incubated for 1 hourand washed 3 times with 1×PBS. TMB substrate solution would then beadded and the reaction stopped by addition of stop solution. OD wouldmeasured at 450 nm to produce a data curve depicting the relationshipbetween OD and MVP concentration. OD₄₅₀ results could resemble the datain Table 3 below.

TABLE 3 OD₄₅₀ measurements from XMuLV MVP removal study DilutionConcentration (MVP/ml) OD₄₅₀ 1 × 10⁹ 1.47 1 × 10⁷ 0.9 1 × 10⁵ 0.56 1 ×10³ 0.33 0 (process solution control) 0.01

The amount of XMuLV MVP removed from processing through the parvovirusfilter could be quantified empirically. The 1 ml samples of processsolution (after MVP addition) and process collection would be subjectedto the same ELISA method described for the serial dilution samplesabove. The OD₄₅₀ results would be plugged into an equation which bestsfits the data from Table 3 to predict the amounts of MVP in solutionprior to filtering and after filtering. From this data, the XMuLV MVPLRV could be calculated as described in Example 6 and according tomethods common in the art for quantifying virus removal.

1-29. (canceled)
 30. A kit comprising: a. at least one containercomprising a Mock Viral Particle (MVP), wherein the MVP is anon-infectious, non-replicating assembled unit comprising a viral capsidor envelope protein and wherein said MVP physiochemically resembles avirus to be evaluated; and b. instructions for performing a method ofquantifying the amount of MVP removed from a process solution, whereinsaid method comprises: i. adding the MVP to the process solution,wherein said process solution comprises a biologic of interest selectedfrom an antibody, non-antibody protein, vaccine, nucleic acid product,blood or plasma derivative; ii. processing the process solution througha purification technique to generate a process collection; and iii.quantifying the amount of MVP removed from the process solution bycomparing the quantity of MVP in the process solution to the quantity ofMVP in the process collection.
 31. The kit of claim 30, wherein saidprocess solution is produced from a cell culture process or afermentation process.
 32. The kit of claim 31, wherein said cell cultureprocess or fermentation process utilizes human cells, animal cells,plant cells, insect cells, hybridomas cells, yeast cell, or bacterialcells.
 33. The kit of claim 30, wherein the purification technique is achromatography, filtration, ultrafiltration, centrifugation, or viralinactivation technique.
 34. The kit of claim 30, wherein the quantity ofMVP in the process solution is greater than the quantity of MVP in theprocess collection.
 35. The kit of claim 30, wherein the MVP comprises aviral capsid and an envelope protein.
 36. The kit of claim 35, whereinsaid viral capsid or envelope protein is produced in a bacterium, yeast,plant, insect cell, animal or human cell.
 37. The kit of claim 35,wherein viral capsid or envelope protein is derived from a Parvoviridaeor Retroviridae source.
 38. The kit of claim 30, wherein said MVPcontains in vitro nucleic acid.
 39. The kit of claim 30, wherein themethod of quantifying the amount of MVP removed utilizes ELISA, FOR,nanoimaging, fluorescence, enzymatic, microscopy, spectrophotometry,transmission electron microscopy (TEM), or western blot analysestechniques.
 40. The kit of claim 30, wherein the method of quantifyingthe amount of MVP removed utilizes an antibody capable of binding to acapsid protein epitope or an envelope protein epitope present on thesurface of the MVP.
 41. The kit of claim 30, wherein the method ofquantifying the amount of MVP removed utilizes a primer capable ofbinding to an in vitro nucleic acid sequence contained within the MVP.42. The kit of claim 30, wherein more than one different species of MVPis added to the process solution.
 43. The kit of claim 30, wherein saidmethod comprises determining the quantity of MVP in an aliquot of theprocess solution or the collection solution.
 44. The kit of claim 30wherein the quantity of MVP in more than one collection solution isdetermined.
 45. The kit of claim 30, wherein the kit further comprisesprimers capable of binding to an in vitro nucleic acid sequencecontained within the MVP.
 46. The kit of claim 30, wherein the kitfurther comprises a molecule capable of binding to the MVP.
 47. The kitof claim 46, wherein the molecule is conjugated to an enzyme.
 48. Thekit of claim46, wherein the molecule is an antibody.
 49. The kit ofclaim 48, wherein the antibody is conjugated to an enzyme.
 50. The kitof claim 46, wherein the molecule capable of binding to the MVP furthercomprises a nucleic acid molecule.
 51. The kit of claim 50, wherein thekit further comprises a set of primers capable of binding to the nucleicacid molecule.
 52. The kit of claim 50, wherein said molecule capable ofbinding to the MVP that further comprises the nucleic acid molecule isan antibody.
 53. The kit of claim 46, wherein said kit further comprisesa second molecule capable of binding to said molecule capable of bindingto the MVP.
 54. The kit of claim 53, wherein said second molecule isconjugated to an enzyme.
 55. The kit of claim53, wherein said secondmolecule is an antibody.
 56. The kit of claim 55, wherein said antibodyis conjugated to an enzyme.
 57. The kit of claim 30, wherein the kitfurther comprises a plate.
 58. The kit of claim 57, wherein said platecomprises an immobilized molecule capable of binding to the MVP.
 59. Thekit of claim 58, wherein said molecule is an antibody.
 60. The kit ofclaim 57, wherein the kit further comprises a molecule capable ofbinding to the MVP.
 61. The kit of claim60, wherein the molecule is anantibody.
 62. The kit of claim 61, wherein the antibody is conjugated toan enzyme.
 63. The kit of claim 60, wherein the molecule capable ofbinding to the MVP further comprises a nucleic acid molecule.
 64. Thekit of claim 63, wherein the kit further comprises a set of primerscapable of binding to the nucleic acid molecule.
 65. The kit of claim63, wherein said molecule capable of binding to the MVP that furthercomprises a nucleic acid molecule is an antibody.
 66. The kit of claim60, wherein said kit further comprises a second molecule capable ofbinding to said molecule capable of binding to the MVP.
 67. The kit ofclaim 66, wherein said second molecule is conjugated to an enzyme. 68.The kit of claim 66, wherein said second molecule is an antibody. 69.The kit of claim 68, wherein said antibody is conjugated to an enzyme.